Fig. 5.

VCP interacts with HMGB1 in HCC cells. A The representative MS/MS spectrum of HMGB1 and the amino acid sequence was identified as KHPDASVNFSEFSK from mass differences in the y and b fragment ions series. B The protein sequence of HMGB1, the matched peptides are labeled in red bold letters. C MHCC-LM3 cells were treated with exogenous VCP and immunoprecipitation was performed. The HMGB1 protein shown by the arrow was observed by Coomassie staining after running SDS-PAGE. D–F GST pull-down assay indicated that VCP could be fused (D) and purified (E) in vitro, and the purified GST-tagged VCP pulls down HMGB1 in MHCC-LM3 cells (F). G, H The CoIP was operated to validate the VCP-HMGB1 interaction in Huh7 and MHCC-LM3 cells. I, J The colocalization of VCP and HMGB1 in HCC cells was observed by confocal microscopy. SDS-PAGE sodium dodecyl sulfate–polyacrylamide gel electrophoresis, MS/MS tandem mass spectrometry, CoIP co-immunoprecipitation